A. Factor H
Factor H, a 150-kDa protein, is a key regulatory braking mechanism in normal and alternate complement-mediated cell lysis. It dissociates and thereby inactivates the assembled C3 convertase, serves as an essential accelerator of Factor I-mediated cleavage of C3b to iC3b, and sterically inhibits C5 binding to C3b (a prerequisite step for terminal pathway activation). The salient structural features of Factor H include 20 short consensus repeats (SCRs) that contain four cysteine residues forming two disulfide bonds per repeat. In addition, each SCR contains at least one conserved tryptophan residue per repeat, and Factor H is known to interact with several sialic acid-containing proteins.
B. Molecular Characterization of BSP, A Member of the SIBLINGS Protein Family
Bone sialoprotein (BSP), also briefly known as BSPII, is a phosphorylated and sulfated glycoprotein that is associated with most normal and many pathological mineralized matrices. It is a small (approximate Mr=75,000) integrin-binding protein that supports cell attachment in vitro through both RGD-dependent and RGD-independent mechanisms and has a high affinity for hydroxyapatite. BSP is a member of the family of small integrin-binding ligand, N-linked glycoproteins (SIBLINGS) family that also includes osteopontin (OPN), dentin matrix protein (DMP1) and dentin sialophosphoprotein (DSPP). All have similar gene structures and are clustered on human chromosome 4.
Bone sialoprotein (BSP) constitutes about 10–15% of the non-collagenous proteins found in the mineralized compartment of young bone (Fisher et el., J. Biol. Chem. 258:12723–727, 1983). Immunolocalization and in situ studies have shown BSP to be produced by osteoblasts, osteocytes, and osteoclasts (the multinucleated cells that resorb bone) (Bianco et al., Calcif. Tissue Int. 49:421–426, 1991). The areas richest in BSP are the collagen-poor matrix found between areas of new bone, where bone is developing or turning over. Outside of bone, BSP has been found in other mineralized tissues, such as dentin (Fisher et al., J. Biol. Chem. 258:12723–727. 1983), cementum (MacNeil et al., J. Bone Min. Res. 9:1597–1606, 1994), and calcifying cartilage of the growth plate (Bianco et al., Calcif. Tissue Int. 49:421–426, 1991). Trophoblasts of the developing placenta also express high levels of BSP (Bianco et al., Calcified Tissue International, 49:421–426, 1991). While placental tissue is not usually considered to be a mineralized tissue, late term human placentas have hydroxyapatite crystals associated with the aging trophoblasts.
Using human BSP as a model (Fisher et al., J. Biol. Chem. 265:2347–2351, 1990), the protein is first made as a 317 amino acid, 35,000 Da protein. A 16 amino acid leader peptide is removed during synthesis. BSP has no disulfide bonds and it is nearly uniformly hydrophilic along its length, indicating that the protein is likely to be an extended rod in solution. There are three regions particularly rich in glutamic acids residues (“polyglutamic acid domains”) that have long been thought to govern the high affinity of this protein for hydroxyapatite. Recent work with recombinant fragments, however, shows that BSP's ability to bind strongly to apatite is found throughout its length (Stubbs et al., Bone Miner. Res. 12:1210–1222, 1997).
Human BSP contains four consensus sequences for N-inked oligosaccharides, three of which are conserved for all mammalian species known to date. These N-linked and the many O-linked oligosaccharides make up approximately 50% of the mass of BSP as it is secreted into the human bone matrix (Fisher et al., J. Biol. Chem. 258:12723–727, 1983). Tyrosine sulfation and serine/threonine phosphorylation make up the remainder of the known post translational modifications. There are three tyrosine-rich domains in BSP, the last two of which flank the RGD domain and are subject to sulfation. The presence or absence of the sulfate groups does not appear to change the ability of fibroblasts to attach in a simple in vitro assay (Mintz et al., J. Biol. Chem. 269:4845–4852, 1994).
The cDNA BSP sequences for rat (Oldberg et al., J. Biol. Chem. 263:19430–19432, 1988), human (Fisher et al., J. Biol. Chem. 265:2347–2351, 1990), mouse (Young et al., Mamm. Genome 5:108–111, 1994), cow (Chenu et al., J. Bone Miner. Res. 9:417–421, 1994), hamster (Sasaguri et al., Direct submission to GenBank, Accession number U65889, 1996) and chicken (Yang et al., J. Bone Miner. Res. 10:632–440, 1995) have been published. The human (Kerr, J. M., Fisher, L. W., Termine, J. D., Wang, M. G., McBride, O. W. and Young, M. F. Genomics 17:408–415, 1993) and chicken (Yang, R. and Gerstenfeld, L. C., J. Cell. Biochem. 64:77–93, 1997) genes have also been published. The human IBSP gene maps very close to two other members of this family, within 340 kb of SPPI (osteopontin), and within 150 kb of DMP1 with the order being: cen-DMP1-IBSP-SPPI-tel on chromosome 4 (Aplin et al., Genomics 30:347–349, 1995; Crosby et al., Genome 7:149–151, 1996). Mouse Tbsp is on the homologous region of chromosome 5 at 56.0 (Young et al., Mamm. Genome 5:108–111, 1994). Other members of the family are also encoded on chromosome 4, for example dentin phosphoprotein and dentin sialoprotein (DSPP) are cleavage products expressed from a single transcript coded by a gene on human chromosome 4 (MacDougall et al., J. Biol. Chem. 272(2):835, 1997).
C. Detection of Human BSP
The detection of human BSP in biological samples has been accomplished using polyclonal antibodies directed towards denatured whole BSP, non-denatured whole BSP, or synthetic fragments of BSP. Certain tumors have been found to ectopically express BSP. For example, BSP has been found to be expressed by breast cancer tissue, prostate cancer tissue, lung cancer tissue and thyroid cancer tissue (Bellahcene et al., Cancer Research 54:823–826, 1994; Bellahcene et al., Calcif. Tissue Int. 61:183–188, 1998; Bellahcene et al., Calcif. Tissue Int. 61:183–188, 1997; and Bellahcene et al., Thyroid 8:637–641, 1998 respectively). Additional studies have shown that BSP mRNA levels are increased in human breast cancer tissue as well as cell lines derived from breast cancer tissue (Bellachcene et al., Laboratory Investigation 75:203–210, 1996).
The detection of BSP in various tissue samples described above has been accomplished through the use of polyclonal antibodies directed to either whole human BSP (LF-6) or synthetic fragments of human BSP, such as the synthetic fragment comprising amino acids 277–294 (LF-83).
An increased level of BSP in serum has been correlated with the presence of hyperparathyroidism, Paget's disease, multiple myeloma and breast cancer (Seibel, et al., J. Clinical Endocrinology and Metabolism, 81:3289–294, 1996). Elevated levels of serum BSP have also been detected in subjects suffering from rheumatoid arthritis (Mansson et al., J. Clin. Invest. 95:1071–1077, 1995).